Concatenation of cyan and yellow fluorescent proteins for efficient resonance energy transfer

Highly efficient fluorescence resonance energy transfer between cyan(CFP) and yellow fluorescent proteins (YFP), the cyan- and yellow-emitting variants of the Aequorea green fluorescent protein, respectively, was achieved by tightly concatenating the two proteins. After the C-terminus of CFP and the N-terminus of YFP were truncated by 11 and 5 amino acids, respectively, the proteins were fused through a leucine-glutamate dipeptide. The resulting chimeric protein, which we called Cy11.5, exhibited a simple emission spectrum that peaked at 527 nm when the protein was excited at 436 nm. The time-resolved emission of Cy11.5 was measured using a streak camera. After excitation of Cy11.5 with a 400 nm ultrashort pulse, a fast decay of the CFP emission and a concomitant rise of the YFP emission were observed with a lifetime of 66 ps. By contrast, the emission from CFP alone showed a decay component with a lifetime of 2.9 ns. We concluded that in fully folded Cy11.5 molecules, intramolecular FRET occurred with an efficiency of 98%. Importantly, most Cy11.5 molecules were properly folded, and the protein was highly resistant to all of the tested proteases. In living cells, therefore, Cy11.5 behaved as a single fluorescent protein with a broad excitation spectrum. Moreover, Cy11.5 was used as an optical highlighter after photobleaching of YFP. When HeLa cells expressing Cy11.5 were irradiated at 514.5 nm, a 10-fold increase in the 475 nm fluorescence intensity was observed. These features make Cy11.5 useful as an optical highlighter and a new-colored fluorescent protein for multicolor imaging.     

Resonance energy transfer between green fluorescent protein variants: complexities revealed with myosin fusion proteins

Green fluorescent protein and its variants are frequently used as F?rster (fluorescence) resonance energy transfer (FRET) pairs to determine the proximity of protein domains. We prepared fusion proteins comprising yellow fluorescent protein-Dictyostelium myosin II motor domain-cyan fluorescent protein (YFP-myosin-CFP) and compared their FRET properties with an existing construct (GFP-myosin-BFP), containing a green fluorescent protein acceptor and blue fluorescent protein donor [Suzuki, Y., Yasunaga, T., Ohkura, R., Wakabayashi, T. and Sutoh, K. (1998) Nature 396, 380-383]. The latter construct showed an apparent 40% reduction in acceptor fluorescence on ATP addition, when excited via the donor, compared with the YFP-myosin-CFP constructs which showed a small increase (相似文献   

Excited-state proton transfer of equilenin and dihydroequilenin: interaction with bilayer vesicles

The two-state excited-state proton-transfer process for d-equilenin [d-3-hydroxyestra-1,3,-5(10),6,8-pentaen-17-one] and dihydroequilenin is found to depend both on pH and on proton acceptor concentration. Both the protonated and deprotonated forms of the excited molecule are fluorescent. As is the case for 2-naphthol, the excited-state pKa (pKa*) is substantially lower than the ground-state pKa. Fluorescence decay studies have been performed as a function of emission wavelength in aqueous solutions at pH 6.9 in the presence of acetate anion (0.1 M). At this pH, both back-reaction from the excited-state and ground-state heterogeneity are minimal. A monoexponential decay is found in the blue region of the spectrum and a biexponential decay on the red edge. The lifetimes measured across both regions are constant, with a negative preexponential term, characteristic of an excited-state reaction, evident at longer wavelengths. Decay-associated spectra (DAS), the preexponential terms associated with the measured lifetimes, have been acquired for these aqueous solutions. Equilenin and dihydroequilenin are found to adsorb to dimyristoyllecithin (DML) vesicles. Rates for excited-state proton transfer are greatly reduced when dihydroequilenin adsorbs to vesicles. The accessibility of the bound probe to acetate as a proton acceptor depends on the cholesterol content of the vesicles.     

Spectrally and spatially resolved fluorescence lifetime imaging in living cells: TRPV4-microfilament interactions

Time- and space-correlated single photon counting method has been used to demonstrate the interactions of cation channel "transient receptor potential vanilloid 4" (TRPV4) and microfilaments. Living cells co-expressing TRPV4-CFP and actin-YFP, when excited for the donor molecules (CFP) exhibited an emission peak at 527 nm and decrease of the lifetime in the wavelength band 460-490 nm; corresponding to resonance energy transfer to YFP. CFP fluorescence decay was fitted best by a dual mode decay model. Considering the average lifetime of the donor, both in the presence and absence of acceptor yielded an apparent FRET efficiency of approximately 20%. This is rather high placing the minimum distance of chromophores in the two fluorescent proteins in the range of 4 nm. Thus, this study shows for the first time that TRPV4 and actin intimately associate within living cells. The significance of this finding for cell volume regulation is highlighted.     

APPL Proteins FRET at the BAR: Direct Observation of APPL1 and APPL2 BAR Domain-Mediated Interactions on Cell Membranes Using FRET Microscopy

Background

Human APPL1 and APPL2 are homologous RAB5 effectors whose binding partners include a diverse set of transmembrane receptors, signaling proteins, and phosphoinositides. APPL proteins associate dynamically with endosomal membranes and are proposed to function in endosome-mediated signaling pathways linking the cell surface to the cell nucleus. APPL proteins contain an N-terminal Bin/Amphiphysin/Rvs (BAR) domain, a central pleckstrin homology (PH) domain, and a C-terminal phosphotyrosine binding (PTB) domain. Previous structural and biochemical studies have shown that the APPL BAR domains mediate homotypic and heterotypic APPL-APPL interactions and that the APPL1 BAR domain forms crescent-shaped dimers. Although previous studies have shown that APPL minimal BAR domains associate with curved cell membranes, direct interaction between APPL BAR domains on cell membranes in vivo has not been reported.

Methodology

Herein, we used a laser-scanning confocal microscope equipped with a spectral detector to carry out fluorescence resonance energy transfer (FRET) experiments with cyan fluorescent protein/yellow fluorescent protein (CFP/YFP) FRET donor/acceptor pairs to examine interactions between APPL minimal BAR domains at the subcellular level. This comprehensive approach enabled us to evaluate FRET levels in a single cell using three methods: sensitized emission, standard acceptor photobleaching, and sequential acceptor photobleaching. We also analyzed emission spectra to address an outstanding controversy regarding the use of CFP donor/YFP acceptor pairs in FRET acceptor photobleaching experiments, based on reports that photobleaching of YFP converts it into a CFP-like species.

Conclusions

All three methods consistently showed significant FRET between APPL minimal BAR domain FRET pairs, indicating that they interact directly in a homotypic (i.e., APPL1-APPL1 and APPL2-APPL2) and heterotypic (i.e., APPL1-APPL2) manner on curved cell membranes. Furthermore, the results of our experiments did not show photoconversion of YFP into a CFP-like species following photobleaching, supporting the use of CFP donor/YFP acceptor FRET pairs in acceptor photobleaching studies.     

Ultrafast excited-state dynamics in the green fluorescent protein variant S65T/H148D. 1. Mutagenesis and structural studies

Wild type green fluorescent protein (wt-GFP) and the variant S65T/H148D each exhibit two absorption bands, A and B, which are associated with the protonated and deprotonated chromophores, respectively. Excitation of either band leads to green emission. In wt-GFP, excitation of band A ( approximately 395 nm) leads to green emission with a rise time of 10-15 ps, due to excited-state proton transfer (ESPT) from the chromophore hydroxyl group to an acceptor. This process produces an anionic excited-state intermediate I* that subsequently emits a green photon. In the variant S65T/H148D, the A band absorbance maximum is red-shifted to approximately 415 nm, and as detailed in the accompanying papers, when the A band is excited, green fluorescence appears with a rise time shorter than the instrument time resolution ( approximately 170 fs). On the basis of the steady-state spectroscopy and high-resolution crystal structures of several variants described herein, it is proposed that in S65T/H148D, the red shift of absorption band A and the ultrafast appearance of green fluorescence upon excitation of band A are due to a very short (相似文献   

Molecular mechanism of a green-shifted, pH-dependent red fluorescent protein mKate variant

Fluorescent proteins that can switch between distinct colors have contributed significantly to modern biomedical imaging technologies and molecular cell biology. Here we report the identification and biochemical analysis of a green-shifted red fluorescent protein variant GmKate, produced by the introduction of two mutations into mKate. Although the mutations decrease the overall brightness of the protein, GmKate is subject to pH-dependent, reversible green-to-red color conversion. At physiological pH, GmKate absorbs blue light (445 nm) and emits green fluorescence (525 nm). At pH above 9.0, GmKate absorbs 598 nm light and emits 646 nm, far-red fluorescence, similar to its sequence homolog mNeptune. Based on optical spectra and crystal structures of GmKate in its green and red states, the reversible color transition is attributed to the different protonation states of the cis-chromophore, an interpretation that was confirmed by quantum chemical calculations. Crystal structures reveal potential hydrogen bond networks around the chromophore that may facilitate the protonation switch, and indicate a molecular basis for the unusual bathochromic shift observed at high pH. This study provides mechanistic insights into the color tuning of mKate variants, which may aid the development of green-to-red color-convertible fluorescent sensors, and suggests GmKate as a prototype of genetically encoded pH sensors for biological studies.     

Phototransduction by vertebrate ultraviolet visual pigments: protonation of the retinylidene Schiff base following photobleaching

The photochemical and subsequent thermal reactions of the mouse short-wavelength visual pigment (MUV) were studied by using cryogenic UV-visible and FTIR difference spectroscopy. Upon illumination at 75 K, MUV forms a batho intermediate (lambda(max) approximately 380 nm). The batho intermediate thermally decays to the lumi intermediate (lambda(max) approximately 440 nm) via a slightly blue-shifted intermediate not observed in other photobleaching pathways, BL (lambda(max) approximately 375 nm), at temperatures greater than 180 K. The lumi intermediate has a significantly red-shifted absorption maximum at 440 nm, suggesting that the retinylidene Schiff base in this intermediate is protonated. The lumi intermediate decays to an even more red-shifted meta I intermediate (lambda(max) approximately 480 nm) which in turn decays to meta II (lambda(max) approximately 380 nm) at 248 K and above. Differential FTIR analysis of the 1100-1500 cm(-1) region reveals an integral absorptivity that is more than 3 times smaller than observed in rhodopsin and VCOP. These results are consistent with an unprotonated Schiff base chromophore. We conclude that the MUV-visual pigment possesses an unprotonated retinylidene Schiff base in the dark state, and undergoes a protonation event during the photobleaching cascade.     

Ultrafast excited-state dynamics in the green fluorescent protein variant S65T/H148D. 2. Unusual photophysical properties

In the preceding accompanying paper [Shu, X., et al. (2007) Biochemistry 46, 12005-12013], the 1.5 A resolution crystal structure of green fluorescent protein (GFP) variant S65T/H148D is presented, and the possible consequences of an unusual short hydrogen bond (相似文献   

A ratiometric fluorescent sensor for Zn2+ based on internal charge transfer (ICT)

A ratiometric fluorescent chemosensor 5 was designed and synthesized based on internal charge transfer (ICT). The indicator absorbs and emits light in the visible wavelength range. In acetonitrile, blue shifts in fluorescent emission upon zinc binding are due to the formation of a 1:2 metal/ligand complex, which induced a fluorescent emission at 616 nm at the expense of the fluorescent emission at 672 nm.     

Crystallographic and energetic analysis of binding of selected anions to the yellow variants of green fluorescent protein

The fluorescence emission of yellow fluorescent proteins (YFPs) has been shown to respond rapidly and reversibly to changes in the concentration of some small anions such as halides; this allows for the use of YFPs as genetically encodable Cl(-) sensors that may be targeted to specific organelles in living cells. Fluorescence is suppressed due to protonation of the chromophore upon anion binding, with a stronger level of interaction at low pH values. At pH 6.0, the apparent dissociation constant (K(app)) for Cl(-) is 32 mM for YFP and 22 mM for YFP-H148Q, whereas at pH 7.5, K(app) is 777 mM and 154 mM, respectively. In the cytosol, YFP-H148Q appears most promising as a halide sensor due to its high degree of sensitivity towards I(-) (K(app)=23 mM at pH 7.5). To aid in the design of variants with improved levels of specificity and affinity for Cl(-), we solved apo and I(-)-bound crystal structures of YFP-H148Q to 2.1 A resolution. The halide-binding site is found near van der Waals contact with the chromophore imidazolinone oxygen atom, in a small buried cavity adjacent to Arg96, which provides electrostatic stabilization. The halide ion is hydrogen bonded to the phenol group of T203Y, consistent with a mutational analysis that indicates that T203Y is indispensible for tight binding. A series of conformational changes occurs in the amphiphilic site upon anion binding, which appear to be propagated to the beta-bulge region around residue 148 on the protein surface. Anion binding raises the chromophore pK(a) values, since delocalization of the phenolate negative charge over the chromophore skeleton is suppressed. Extraction of microscopic binding constants for the linked equilibrium between anion and proton binding indicates that anion selectivity by YFP is related to hydration forces. Specific suggestions to improve Cl(-) binding to YFP-H148Q based on size and hydration energy are proposed.     

Reversible photobleaching of enhanced green fluorescent proteins

Color variants of green fluorescent protein (GFP) are increasingly used for multicolor imaging, fluorescence resonance energy transfer (FRET), and fluorescence recovery after photobleaching (FRAP). Here we show that experimental settings commonly used in these imaging experiments may induce an as yet uncharacterized reversible photobleaching of fluorescent proteins, which is more pronounced at acidic pH. Whereas the reversible photobleaching spectrum of eCFP corresponds to its absorption spectrum, reversible photobleaching spectra of yellow variants resemble absorption spectra of their protonated states. Fluorescence intensities recover spontaneously with time constants of 25-58 s. The recovery of eCFP can be further accelerated by illumination. The resulting steady-state fluorescence reflects a variable equilibrium between reversible photobleaching, spontaneous recovery, and light-induced recovery. These processes can cause significant artifacts in commonly applied imaging techniques, photobleach-based FRET determinations, and FRAP assays.     

Molecular basis for pH sensitivity and proton transfer in green fluorescent protein: protonation and conformational substates from electrostatic calculations.

We performed a theoretical study to elucidate the coupling between protonation states and orientation of protein dipoles and buried water molecules in green fluorescent protein, a versatile biosensor for protein targeting. It is shown that the ionization equilibria of the wild-type green fluorescent protein-fluorophore and the internal proton-binding site E222 are mutually interdependent. Two acid-base transitions of the fluorophore occur in the presence of neutral (physiologic pH) and ionized (pH > 12) E222, respectively. In the pH-range from approximately 8 to approximately 11 ionized and neutral sites are present in constant ratio, linked by internal proton transfer. The results indicate that modulation of the internal proton sharing by structural fluctuations or chemical variations of aligning residues T203 and S65 cause drastic changes of the neutral/anionic ratio-despite similar physiologic fluorophore pK(a) s. Moreover, we find that dipolar heterogeneities in the internal hydrogen-bond network lead to distributed driving forces for excited-state proton transfer. A molecular model for the unrelaxed surrounding after deprotonation is discussed in relation to pathways providing fast ground-state recovery or slow stabilization of the anion. The calculated total free energy for excited-state deprotonation ( approximately 19 k(B)T) and ground-state reprotonation ( approximately 2 k(B)T) is in accordance with absorption and emission data (相似文献   

Scavenger receptor class B, type I (SR-BI) homo-dimerizes via its C-terminal region: fluorescence resonance energy transfer analysis

Expression of the scavenger receptor class B, type I (SR-BI) receptor facilitates high density lipoprotein cholesterol transport and correlates with protection against atherosclerosis. Studies have shown that SR-BI self-associates, but many of the techniques used to characterize SR-BI homo-oligomerization were wrought with the prospect of producing artifacts. Therefore, we employed fluorescence resonance energy transfer (FRET) to visualize SR-BI homo-oligomerization with the benefit of gaining information about its quaternary structure in the absence of typical membrane receptor artifacts. To this end, SR-BI was tagged at the N- or C-termini with either cyan (CFP) or yellow (YFP) fluorescent protein. To test whether SR-BI subunits oligomerize through N-N, N-C or C-C terminal interactions, we co-expressed the appropriate SR-BI fusion protein combinations in COS-7 cells and measured live-cell FRET following acceptor photobleaching. We did not observe FRET with co-transfection of SR-BI with CFP and YFP at the N-termini nor at the N- and C-termini, suggesting that the N-termini are not proximal to each other or to the C-termini. However, FRET was observed with co-transfection of SR-BI with CFP and YFP at the C-termini, suggesting that the C-terminal ends are within 10 nm of each other, consistent with SR-BI dimerization via its C-terminal region.     

The chemical synthesis of N-[1-(2-naphthol)]-phosphatidylethanolamine, a fluorescent phospholipid for excited-state proton transfer studies.

A procedure for the preparation of N-[1-(2-naphthol)]-phosphatidylethanolamine (NAPH-PE) has been developed. The synthesis is based on the Schiff base formation between the NH2 of the phospholipid and the aldehyde moiety of 2-hydroxy-1-naphthaldehyde. Then selective reduction of the imine is used to obtain the stable secondary amine, NAPH-PE. Formation of the intermediate Schiff base and the final product is confirmed by 13C- and 1H-NMR. Similar to free 2-naphthol, the excited-state pKa (pKa*) of its phospholipid derivative appears to be significantly lower than the ground-state pKa. At pH 7.4, the excitation spectrum of NAPH-PE shows no deprotonated species in the ground-state, while the emission spectrum presents a significant contribution of this species. Thus the fluorescent phospholipid exhibits the typical behavior of excited-state proton-transfer probes. NAPH-PE is found to incorporate in dimyristoyllecithin (DML) vesicles. The emission spectrum of the probe inserted in the liposomes is affected by acetate used as a proton acceptor. These properties should also be manifest in other lipid bilayers (e.g., plasma membranes of cells) and used for excited-state proton transfer studies.     

Organization of calcium channel beta1a subunits in triad junctions in skeletal muscle

In skeletal muscle, dihydropyridine receptors (DHPRs) in the plasma membrane interact with the type 1 ryanodine receptor (RyR1) at junctions with the sarcoplasmic reticulum. This interaction organizes junctional DHPRs into groups of four termed tetrads. In addition to the principle alpha1S subunit, the beta1a subunit of the DHPR is also important for the interaction with RyR1. To probe this interaction, we measured fluorescence resonance energy transfer (FRET) of beta1a subunits labeled with cyan fluorescent protein (CFP) and/or yellow fluorescent protein (YFP). Expressed in dysgenic (alpha1S-null) myotubes, YFP-beta1a-CFP and CFP-beta1a-YFP were diffusely distributed in the cytoplasm and highly mobile as indicated by fluorescence recovery after photobleaching. Thus, beta1a does not appear to bind to other cellular proteins in the absence of alpha1S. FRET efficiencies for these cytoplasmic beta1a subunits were approximately 6-7%, consistent with the idea that <10 nm separates the N and C termini. After coexpression with unlabeled alpha1S (in dysgenic or beta1-null myotubes), both constructs produced discrete fluorescent puncta, which correspond to assembled DHPRs in junctions and that did not recover after photobleaching. In beta1-null myotubes, FRET efficiencies of doubly labeled beta1a in puncta were similar to those of the same constructs diffusely distributed in the cytoplasm and appeared to arise intramolecularly, since no FRET was measured when mixtures of singly labeled beta1a (CFP or YFP at the N or C terminus) were expressed in beta1-null myotubes. Thus, DHPRs in tetrads may be arranged such that the N and C termini of adjacent beta1a subunits are located >10 nm from one another.     

A peptide-based, ratiometric biosensor construct for direct fluorescence detection of a protein analyte

We present the design, synthesis, and functional evaluation of peptide-based fluorescent constructs for wavelength-ratiometric biosensing of a protein analyte. The concept was shown using the high-affinity model interaction between the 18 amino acid peptide pTMVP and a recombinant antibody fragment, Fab57P. pTMVP was functionalized in two different positions with 6-bromomethyl-2-(2-furanyl)-3-hydroxychromone, an environmentally sensitive fluorophore with a two-band emission. The equilibrium dissociation constant of the interaction between pTMVP and Fab57P was largely preserved upon labeling. The biosensor ability of the labeled peptide constructs was evaluated in terms of the relative intensity change of the emission bands from the normal (N*) and tautomer (T*) excited-state species of the fluorophore ( I(N*)/I(T*)) upon binding of Fab57P. When the peptide was labeled in the C terminus, the I(N*)/I(T*) ratio changed by 40% upon analyte binding, while labeling close to the residues most important for binding resulted in a construct that completely lacked ratiometric biosensor ability. Integrated biosensor elements for reagentless detection, where peptides and ratiometric fluorophores are combined to ensure robustness in both recognition and signaling, are expected to become an important contribution to the design of future protein quantification assays in immobilized formats.     

One- and two-photon-induced fluorescence from recombinant green fluorescent protein

The fluorescence spectral properties of recombinant green fluorescent protein (rGFP) were examined with one- and two-photon excitations using femtosecond pulses from a Ti:sapphire laser. Intensity-dependent properties of the two-photon-induced fluorescence from rGFP excited by an 800-nm, 100-fs laser beam were reported, and the two-photon excitation cross section of rGFP was measured at 800 nm as about 160 x 10(-50) cm(4)s/photon. The possible excited-state proton transfer between two electronic states at about 400 nm in protonated (RH) species and 478 nm in deprotonated (R(-)) species in rGFP was confirmed by fluorescence and fluorescence excitation anisotropy spectra. A subelectronic state (or vibronic progression) at about 420 nm in RH species was identified, which was relatively stable and not involved in the excited state proton transfer in rGFP upon irradiation.     

Chemical and photophysical mechanism of fluorescence enhancement of 3-quinolineboronic acid upon change of pH and binding with carbohydrates

The free 3-quinolineboronic acid (3-QBA) with the lowest (n-π*) excited singlet is non- or weakly fluorescent while protonated 3-QBA has the lowest (π-π*) excited singlet state and is highly fluorescent. The hybridization of boronic atom or charge transfer from aromatic ring to boronic acid group plays a secondary role in affecting fluorescence intensity. Binding with carbohydrate at a proper acidity, the hybridization of boron atom changes from sp(2) to sp(3) and the nitrogen atom in the quinoline ring is partially protonated, resulting in large enhancement of fluorescence. Meanwhile, the fluorescent lifetime of 3-QBA produces obvious change by binding with carbohydrates. Quinoline boronic acid is an important water-soluble fluorescence sensor for carbohydrate recognition. Both the remarkable changes in intensity and lifetime of 3-QBA can act as working parameters in recognition of carbohydrates at physiological pH.     

DNA labeling in living cells.

BACKGROUND: Live cell fluorescence microscopy experiments often require visualization of the nucleus and the chromatin to determine the nuclear morphology or the localization of nuclear compartments. METHODS: We compared five different DNA dyes, TOPRO-3, TOTO-3, propidium iodide, Hoechst 33258, and DRAQ5, to test their usefulness in live cell experiments with continuous imaging and photobleaching in widefield epifluorescence and confocal laser scanning microscopy. In addition, we compared the DNA stainings with fluorescent histones as an independent fluorescent label to mark chromatin. RESULTS: From the dyes tested, only Hoechst and DRAQ5 could be used to stain DNA in living cells. However, DRAQ5 had several advantages, namely low photobleaching, labeling of the chromatin compartments comparable to that of H2B-GFP fusion proteins, and deep red excitation/emission compatible with available genetically encoded fluorescent proteins such as C/G/YFP or mRFP. CONCLUSIONS: The DNA dye DRAQ5 is well suited for chromatin visualization in living cells and can easily be combined with other fluorophores with blue to orange emission.